Cell growth inhibitor containing anti-PepT antibody

ABSTRACT

The present inventors extensively studied and found that an antibody binding to PepT has cytotoxic activity and inhibits cell growth. These results suggest that an antibody binding to PepT, particularly an antibody having a cytotoxic activity, can be used as a cell growth inhibitor, for example, in treating and preventing cancer.

TECHNICAL FIELD

The present invention relates to an antibody binding to PepT and a cellgrowth inhibitor containing the antibody as an effective ingredientthereof.

BACKGROUND ART

Mammalian animals need to take in external sources of nutrition and manytransport proteins are known to exist in their cells. Many peptidetransporters (peptide transport proteins; PepTs) that carry out peptidetransport have been found to date (for example, J. Biol. Chem.,270(12):6456-6463, (1995); Biochim. Biophys. Acta., 1235:461-466,(1995); Mol. Microbiol., Vol. 16, p 825, (1995); Unexamined PublishedJapanese Patent Application No. (JP-A) Hei 6-261761; JP-A Hei 11-172;and U.S. Pat. No. 5,849,525). PepT can be classified into proteins thatimport peptides into cells and proteins that export peptides from cells.They can also be classified according to the different energy sourcesused during transport. Proton-driven PepTs, which carry out transport byutilizing protein gradient, belong to the PTR family (Mol. Microbiol.,Vol. 16, p 825, (1995)). PepTs that carry out transport using ATP in thebody belong to the ABC family (Annu. Rev. Cell. Biol., Vol. 8, p 67,(1992)).

There are reports that PepTs are involved in the transport of not onlysmall-molecule peptides such as dipeptides and tripeptides, but also ofpharmaceutical agents such as β-lactam antibiotics and ACE inhibitors(Ganapathy, Leibach., Curr. Biol. 3, 695-701, (1991); Nakashima et al.,Biochem. Pharm. 33, 3345-3352, (1984); Friedman, Amidon., Pharm. Res.,6, 1043-1047, (1989); Okano et al., J. Biol. Chem., 261, 14130-14134,(1986); Muranushi et al., Pharm. Res., 6, 308-312, (1989); Friedman,Amidon., J. Control. Rel., 13, 141-146, (1990)).

PepT1 and PepT2 are proton-driven PepTs that contribute to theabsorption of proteins and the maintenance of peptidic nitrogen sourcesby uptaking small-molecule peptides into cells. PepT1 and PepT2 are12-transmembrane proteins, comprising 708 and 729 amino acids,respectively (J. Biol. Chem., 270(12):6456-6463, (1995); Biochim.Biophys. Acta., 1235:461-466, (1995); and Terada and Inui, TanpakusitsuKakusan Kouso., Vol. 46, No. 5, (2001)).

There are reports that PepT1 and PepT2 also transport pharmaceuticalssuch as β-lactam antibiotics and bestatin (Saito, H. et al., J.Pharmacol. Exp. Ther., 275, 1631-1637, (1995); Saito, H. et al.,Biochim. Biophys. Acta., 1280, 173-177, (1996); and Terada, T. et al.,J. Pharmacol. Exp. Ther., 281, 1415-1421 (1997)).

PepT1 is mainly expressed in the small intestine and its expression hasbeen confirmed in the kidney and pancreas. Expression of PepT2 has beenconfirmed in the kidney, brain, lung, and spleen. PepT1 and PepT2 havebeen reported to be localized in the brush border membrane of intestinaland renal epithelial cells (Ogihara, H. et al., Biochem. Biophys. Res.Commun. 220, 848-852, (1996); Takahashi, K. et al., J. Pharmacol. Exp.Ther., 286, 1037-1042 (1998); Hong, S. et al., Am. J. Physiol. Renal.Physiol., 276, F658-F665 (1999); and Terada and Inui, TanpakusitsuKakusan Kouso., Vol. 46, No. 5, (2001)).

Furthermore, overexpression of PepT1 in the cell membrane of humanpancreatic duct carcinoma cell lines (Cancer Res., 58, 519-525, (1998))and the expression of PepT2 mRNA in human pancreatic duct carcinoma celllines (Millennium World Congress of Pharmaceutical Sciences, (2000))have been reported. However, the involvement of PepT1 and PepT2 incancer cell growth was unclear and no discussion had been made as towhether PepT1 and PepT2 when used as target antigens against antibodieswill affect cancer cell proliferation.

DISCLOSURE OF THE INVENTION

The present invention has been made in view of the above observations,aiming at providing an antibody binding to PepT and effectivelyinhibiting cell growth. Furthermore, this invention also aims atproviding a cell growth inhibitor that contains the antibody as aneffective ingredient.

The present inventors extensively studied and found that an antibodybinding to PepT has cytotoxic activity and inhibits cell growth. Theseresults suggest that an antibody binding to PepT, particularly anantibody having cytotoxic activity, can be used as a cell growthinhibitor.

Specifically, the present invention provides:

[1] a cell growth inhibitor comprising an antibody binding to PepT as aneffective ingredient;

[2] the cell growth inhibitor according to [1], wherein the antibodybinding to PepT has a cytotoxic activity;

[3] the cell growth inhibitor according to [2], wherein the cytotoxicactivity is an antibody-dependent cell-mediated cytotoxic (ADCC)activity;

[4] the cell growth inhibitor according to [2], wherein the cytotoxicactivity is a complement-dependent cytotoxic (CDC) activity;

[5] the cell growth inhibitor according to any one of [1] to [4],wherein the PepT is PepT1;

[6] the cell growth inhibitor according to any one of [1] to [5],wherein the cell growth inhibitor inhibits the growth of a cancer cell;

[7] the cell growth inhibitor according to [6], wherein the cancer cellis a pancreatic cancer cell;

[8] a method for causing toxicity to a cell, wherein the methodcomprises the step of administering an antibody binding to PepT;

[9] an antibody binding to PepT and having a cytotoxic activity;

[10] the antibody according to [9], wherein the cytotoxic activity is anantibody-dependent cell-mediated cytotoxic (ADCC) activity;

[11] the antibody according to [9], wherein the cytotoxic activity is acomplement-dependent cytotoxic (CDC) activity;

[12] the antibody according to [9], wherein the antibody specificallybinds to an extracellular region of PepT;

[13] the antibody according to [9], wherein the PepT is derived fromhuman; and

[14] the antibody according to any one of [9] to [13], wherein the PepTis PepT1.

Firstly, the present invention provides a cell growth inhibitorcontaining an antibody binding to PepT as an effective ingredient.

In this invention, the phrase “containing an antibody binding to PepT asan effective ingredient” means containing an anti-PepT antibody as amajor active ingredient, but it is not intended to limit the anti-PepTantibody content.

There is no particular limitation in the type of an antibody containedin the cell growth inhibitor of this invention so long as it is capableof binding to PepT. In one preferred embodiment, the antibodyspecifically binds to PepT. In another preferred embodiment, theantibody has a cytotoxic activity.

A cytotoxic activity in this invention includes, the antibody-dependentcell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity(CDC). In the present invention, the CDC activity means a cytotoxicactivity mediated by a complement system. The ADCC activity in thepresent invention means an activity to cause cytotoxicity to a targetcell when a specific antibody binds to a surface antigen of the targetcell, following which an Fcγ receptor-containing cell (such asimmunocyte) binds to the Fc moiety of the antibody via the Fcγ receptor.

Whether an anti-PepT antibody has either ADCC activity or CDC activitycan be determined by methods well known in the art (for example, Currentprotocols in Immunology, Chapter 7. Immunologic studies in humans,Editor, John E. Coligan et al., John Wiley & Sons, Inc. (1993)).

Specifically, effector cells, complement solution, and target cells areprepared first.

(1) Preparation of Effector Cells

Spleen is excised from a CBA/N mouse or such to isolate spleen cells inRPMI1640 medium (GIBCO). After washing cells with the same mediumcontaining 10% fetal bovine serum (FBS) (HyClone), the cell density isadjusted to 5×10⁶ cells/ml for preparing the effector cells.

(2) Preparation of Complement Solution

Baby Rabbit Complement (CEDARLANE) is diluted 10-fold in a mediumcontaining 10% FBS (GIBCO) to prepare the complement solution.

(3) Preparation of Target Cells

Pancreatic cancer cell line (e.g., AsPc-1 or Capan-2) cells areradiolabeled by incubation with 0.2 mCi ⁵¹Cr-sodium chromate (AmershamPharmacia Biotech) in DMEM medium containing 10% FBS at 37° C. for 1 h.Then, cells are washed three times with RPMI1640 medium containing 10%FBS, and adjusted to the cell density of 2×10⁵ cells/ml to prepare thetarget cells.

Then, ADCC or CDC activity is measured. For ADCC activity, the targetcells and anti-PepT antibodies are added (50 μl each/well) into a96-well U-bottomed plate (Beckton Dickinson), and allowed to react onice for 15 min. After the reaction, effector cells (100 μl) are added toeach well, and the plate is incubated in a carbon dioxide gas incubatorfor 4 h. The final concentration of the antibody is set at 0 μg or 10μg/ml. After incubation, the supernatant (100 μl) is collected and theradioactivity is measured via a gamma counter (COBRAIIAUTO-GMMA, MODELD5005, Packard Instrument Company). Cytotoxic activity (%) can becalculated by the formula:(A−C)/(B−C)×100wherein A represents the radioactivity (cpm) of each sample; Brepresents the radioactivity of a sample comprising 1% NP-40 (Nacalai);and C represents the radioactivity of a sample comprising only thetarget cells.

On the other hand, for CDC activity, the target cells and anti-PepTantibodies are added (50 μl each/well) into a 96-well flat-bottomedplate (Becton Dickinson), and allowed to react on ice for 15 min. Then,the complement solution (100 μl) is added to each well, and incubated ina carbon dioxide gas incubator for 4 h. The final concentration of theantibody is set at 0 μg or 3 μg/ml. After the incubation, thesupernatant (100 μl) is recovered to be measured for its radioactivitywith a gamma counter. The cytotoxic activity can be calculated in thesame manner as the ADCC activity assay.

There are no particular limitations on the antibodies comprised by thecell growth inhibitors of the present invention, as long as they bind tothe antigen. Mouse antibodies, rat antibodies, rabbit antibodies, sheepantibodies, chimeric antibodies, humanized antibodies, and humanantibodies may be used appropriately. Although the antibodies may beeither polyclonal or monoclonal antibodies, monoclonal antibodies arepreferred from the point of view that they can stably producehomogeneous antibodies. Polyclonal and monoclonal antibodies can beprepared by methods well known to those skilled in the art.

Hybridoma cells that produce monoclonal antibodies can basically beproduced using conventional techniques, described as follows:Specifically, the hybridoma cells can be prepared by (1) conductingimmunization using the desired antigen or cells expressing the desiredantigen, as the sensitizing antigen according to standard immunizationmethods; (2) fusing the obtained immunized cells with conventionalparent cells by normal cell fusion methods; and (3) screening formonoclonal antibody-producing cells (hybridomas) using normal screeningmethods.

There is no particular limitation in the type of sensitizing antigen.For example, when PepT is the human PepT1, the human PepT1 protein,cells expressing said human PepT1 protein, partial peptides of the humanPepT1 (such as ndltdhnhdgtpds (SEQ ID NO: 1), sspgspvtavtddfkq (SEQ IDNO: 2), tddfkqgqrht (SEQ ID NO: 3), apnhyqvvkdglnqkpe (SEQ ID NO: 4),kdglnqkpekgeng (SEQ ID NO: 5), scpevkvfedisant (SEQ ID NO: 6), andksnpyfmsgansqkq (SEQ ID NO: 7)) and such can be used.

Antigens can be prepared according to methods using baculoviruses (e.g.WO 98/46777).

Hybridomas can be produced according to the method of Milstein et al.(Kohler, G. and Milstein, C., Methods Enzymol. (1981) 73: 3-46). Whenthe antigen has low immunogenicity, immunization can be performed bylinking it to a macromolecule with immunogenicity, such as albumin.Recombinant antibodies can also be used, and can be produced by (1)cloning an antibody gene from a hybridoma; (2) incorporating theantibody gene into an appropriate vector; (3) introducing the vectorinto a host; and (4) producing the recombinant antibodies by geneticengineering techniques (see, for example, Carl, A. K. Borrebaeck, James,W. Larrick, THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the UnitedKingdom by MACMILLAN PUBLISHERS LTD, 1990). Specifically, cDNAs of thevariable regions (V regions) of antibodies are synthesized fromhybridoma mRNAs using reverse transcriptase. When DNAs encoding a Vregion of an antibody of interest are obtained, they are linked to DNAsencoding an antibody constant region (C region) of interest, and arethen incorporated into expression vectors. Alternatively, DNAs encodingan antibody V region can be incorporated into expression vectorscomprising DNAs of an antibody C region. The DNAs are incorporated intoexpression vectors such that expression is controlled by expressionregulatory regions such as enhancers and promoters. Host cells are thentransformed with these expression vectors to express the antibodies.

The anti-PepT antibody of this invention may recognize any epitopeexisting on the PepT molecule, without being limited to a particularone. However, because PepT is a twelve-transmembrane protein, theepitope present in the extracellular region is preferably recognized.

In the present invention, recombinant antibodies artificially modifiedto reduce heterologous antigenicity against humans can be used. Examplesinclude chimeric antibodies and humanized antibodies. These modifiedantibodies can be produced using known methods. A chimeric antibody isan antibody comprising the antibody heavy chain and light chain variableregions of a nonhuman mammal such as a mouse, and the antibody heavychain and light chain constant regions of a human. A chimeric antibodycan be obtained by (1) ligating the DNA encoding a variable region of amouse antibody to the DNA encoding a constant region of a humanantibody; (2) incorporating them into an expression vector; and (3)introducing the vector into a host for production of the antibody.

A humanized antibody, which is also called a reshaped human antibody, isobtained by transplanting a complementarity determining region (CDR) ofan antibody of a nonhuman mammal such as a mouse, into the CDR of ahuman antibody. Conventional genetic recombination techniques for thepreparation of such antibodies are known. Specifically, a DNA sequencedesigned to ligate a CDR of a mouse antibody with the framework regions(FRs) of a human antibody is synthesized by PCR, using severaloligonucleotides constructed to comprise overlapping portions at theirends. A humanized antibody can be obtained by (1) ligating the resultingDNA to a DNA that encodes a human antibody constant region; (2)incorporating this into an expression vector; and (3) transfecting thevector into a host to produce the antibody (see, European PatentApplication No. EP 239,400, and International Patent Application No. WO96/02576). Human antibody FRs that are ligated via the CDR are selectedwhere the CDR forms a favorable antigen-binding site. As necessary,amino acids in the framework region of an antibody variable region maybe substituted such that the CDR of a reshaped human antibody forms anappropriate antigen-binding site (Sato, K. et al., Cancer Res. (1993)53, 851-856).

Methods for obtaining human antibodies are also known. For example,desired human antibodies with antigen-binding activity can be obtainedby (1) sensitizing human lymphocytes with antigens of interest or cellsexpressing antigens of interest in vitro; and (2) fusing the sensitizedlymphocytes with human myeloma cells such as U266 (see ExaminedPublished Japanese Patent Application No. (JP-B) Hei 1-59878).Alternatively, the desired human antibody can also be obtained by usingthe desired antigen to immunize a transgenic animal that comprises theentire repertoire of human antibody genes (see International PatentApplication WO 93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO96/34096, and WO 96/33735). Furthermore, techniques to obtain humanantibodies by panning with a human antibody library are known. Forexample, the variable region of a human antibody is expressed as asingle chain antibody (scFv) on the surface of a phage using phagedisplay method, and phages that bind to the antigen can be selected. Byanalyzing the genes of selected phages, the DNA sequences encoding thevariable regions of human antibodies that bind to the antigen can bedetermined. If the DNA sequences of scFvs that bind to the antigen areidentified, appropriate expression vectors containing these sequencescan be constructed, and human antibodies can be obtained. Such methodsare already well known (see WO 92/01047, WO 92/20791, WO 93/06213, WO93/11236, WO 93/19172, WO 95/01438, and WO 95/15388).

When the antibody genes have been isolated and introduced into anappropriate host, hosts and expression vectors can be used inappropriate combination to produce the antibodies. As eukaryotic hostcells, animal cells, plant cells, and fungal cells may be used. Knownanimal cells include: (1) mammalian cells such as CHO, COS, myeloma,baby hamster kidney (BHK), HeLa, and Vero cells; (2) amphibian cellssuch as Xenopus oocytes; or (3) insect cells such as sf9, sf21, and Tn5.Known plant cells include cells derived from the Nicotiana genus such asNicotiana tabacum, which can be callus cultured. Known fungal cellsinclude yeasts such as the Saccharomyces genus, for exampleSaccharomyces cerevisiae, and filamentous fungi such as the Aspergillusgenus, for example Aspergillus niger. Prokaryotic cells can also be usedin production systems that utilize bacterial cells. Known bacterialcells include E. coli and Bacillus subtilis. By transferring theantibody genes of interest into these cells using transformation, andthen culturing the transformed cells in vitro, the antibodies can beobtained.

Furthermore, the antibody may be an antibody fragment or a modifiedantibody thereof, as long as it binds to PepT. For example, the antibodyfragment may be Fab, F (ab′) 2, Fv, single chain Fv (scFv) in which Fvfrom H or L chains are ligated by an appropriate linker, or Diabody.More specifically, the antibody fragment is obtained by (1) treating theantibody with enzymes such as papain and pepsin; (2) transferring itinto an expression vector; and then (3) expressing it in an appropriatehost cell (see, for example, Co, M. S. et al., J. Immunol. (1994) 152,2968-2976; Better, M. & Horwitz, A. H. Methods in Enzymology (1989) 178,476-496, Academic Press, Inc.; Plueckthun, A. & Skerra, A. Methods inEnzymology (1989) 178, 476-496, Academic Press, Inc.; Lamoyi, E.,Methods in Enzymology (1989) 121, 663-669; and Bird, R. E. et al.,TIBTECH (1991) 9, 132-137).

scFv can be obtained by ligating the V regions of the antibody H-chainand L-chain. In the scFv, the V regions of the H chain and L chain areligated via a linker, and preferably via a peptide linker (Huston, J. S.et al., Proc. Natl. Acad. Sci. U.S.A (1988) 85, 5879-5883). The Vregions of the scFv H chain and L chain may be derived from any of theantibodies described herein. The peptide linker used to ligate the Vregions may be any single-chain peptide consisting of 12 to 19 residues.DNA encoding scFv can be amplified by PCR using as a template either thewhole DNA, or a partial DNA encoding a desired DNA, selected from a DNAencoding the H chain or the V region of the H chain of the aboveantibody, and a DNA encoding the L chain or the V region of the L chainof the above antibody; and using a primer pair that defines the twoends. Further amplification can be subsequently conducted using thecombination of DNA encoding the peptide linker portion, and the primerpair that defines both ends of the DNA to be ligated to the H chain andthe L chain respectively. Once DNAs encoding scFvs are constructed,expression vectors containing the DNAs, and hosts transformed by theseexpression vectors, can be obtained according to conventional methods.Furthermore, scFvs can be obtained according to conventional methodsusing the resulting hosts. These antibody fragments can be produced inhosts by obtaining genes encoding the antibody fragments and expressingthem in a manner similar to that outlined above. Antibodies bound tovarious types of molecules, such as polyethylene glycol (PEG), may beused as modified antibodies. Furthermore, antibodies may bind toradioisotopes, chemotherapeutics, and cytotoxic substances such asbacteria-derived toxin. In particular, radiolabeled antibodies areuseful. Such modified antibodies can be obtained by chemicalmodifications of the resulting antibodies. Methods for modifyingantibodies are already established in the art. The term “antibody” inthe present invention also encompasses the above-described antibodies.

Furthermore, the antibody used in the present invention may be abispecific antibody. The bispecific antibody may, have antigen-bindingsites recognizing different epitopes on the PepT molecule, or may haveone antigen-binding site recognizing PepT and the other recognizing acytotoxic substance such as radioactive substance, chemotherapeuticagent, and cell-derived toxin. In this case, it is possible to inhibitthe growth of tumor cells by directly applying the cytotoxic substanceto the cells expressing PepT to specifically damage them. Bispecificantibodies can be prepared by linking HL pairs of two kinds ofantibodies, or obtained by fusing hybridomas that produce differentmonoclonal antibodies to prepare fused cells generating bispecificantibody. Furthermore, the bispecific antibody can be generated by usinggenetic engineering techniques.

Antibodies expressed and produced as described above can be purified byconventional methods for purifying normal proteins. Antibodies can beseparated and purified by, appropriately selecting and/or combiningaffinity columns such as a protein A column, or a chromatography column,filtration, ultrafiltration, salt precipitation, dialysis, and such(Antibodies A Laboratory Manual. Ed Harlow, David Lane, Cold SpringHarbor Laboratory, 1988).

Conventional means can be used to measure the antigen-binding activityof the antibodies (Antibodies A Laboratory Manual. Ed Harlow, DavidLane, Cold Spring Harbor Laboratory, 1988). For example, enzyme linkedimmunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay(RIA), or fluoroimmunoassay may be used.

Furthermore, PepT-binding antibodies contained in the cell growthinhibitors of this invention are not particularly limited, however arepreferably antibodies binding to PepTs which have the transport activityof incorporating peptides into cells using proton motive force. Morepreferably, they are antibodies binding to PepT1 or PepT2, and mostpreferably, they are antibodies binding to PepT1.

The nucleotide and amino acid sequences of PepT1 and PepT2 are alreadyknown (human PepT1: GenBank XM 007063 (J. Biol. Chem.,270(12):6456-6463, (1995)); human PepT2: GenBank XM 002922 (Biochim.Biophys. Acta., 1235:461-466, (1995)); mouse PepT1: GenBank AF 205540(Biochim. Biophys. Acta., 1492:145-154 (2000)); and mouse PepT2: GenBankNM 021301 (Biochim. Biophys. Res. Commun., 276:734-741 (2000))).

Furthermore, a preferred antibody binding to PepT of the presentinvention specifically binds to the extracellular region of PepT. Inthis invention, the phrase “specific binding to the extracellularregion” means that the antibody is able to immunologically distinguishthe extracellular region of PepT from other regions. More specifically,the antibody specifically binding to the extracellular region of PepTonly binds to the extracellular region but not to the intracellularregion and such as well as transmembrane domains. In this invention, apreferred PepT is the human PepT. The human PepT can be not only derivedfrom human but also obtained as a recombinant by expressing the humanPepT in the baculoviral expression system. An immunogen used forobtaining antibody which binds specifically to the extracellular regioncan include, PepT expressed on the membrane such as cytoplasmic andviral membranes, and fragments containing the PepT extracellular region.Furthermore, regardless of the transporter activity, both PepTs with orwithout the transport activity can be used as immunogens. For PepT withthe transporter activity, PepT expressed on the membrane such ascytoplasmic and viral membranes (for example, PepTs expressed on theBa/F3 cell membrane and baculoviral membrane) can be used. For example,since PepT is known to incorporate glycylsarcosine into cells as asubstrate, it is possible to judge whether the PepT has the transportactivity or not by contacting it with [¹⁴C]glycylsarcosine to observethe uptake thereof.

There are no particular limitations as to the cells to be targeted bythe growth inhibitors, but cancer cells such as pancreatic cancer cells,liver cancer cells, lung cancer cells, esophageal cancer cells, breastcancer cells, and colon cancer cells are preferred, and pancreaticcancer cells are especially preferred. Therefore, the cell growthinhibitors of the present invention can be used for the purpose oftreatment and prevention of diseases caused by cell growth, and morespecifically of cancers such as pancreatic cancer.

The cell growth inhibitors of the present invention can be administeredeither orally or parenterally, but are preferably administeredparenterally. Specific examples include injections, nasal formulations,pulmonary formulations, and cutaneous formulations. For example,injections can be administered systemically or locally by intravenousinjection, intramuscular injection, intraperitoneal injection, orsubcutaneous injection. Furthermore, the method of administration can beselected appropriately according to the age and symptoms of the patient.A single dose can be selected, from within the range of 0.0001 mg to1,000 mg per kg body weight. Alternatively, the dose can be selected,from within the range of 0.001 to 100,000 mg/body for each patient.However, the dose of a therapeutic agent of the present invention is notlimited to these examples.

The cell growth inhibitors of the present invention can be formulatedaccording to standard methods (see, for example, Remington'sPharmaceutical Science, latest edition, Mark Publishing Company, Easton,U.S.A), and may comprise pharmaceutically acceptable carriers andadditives. Exemplary carriers include surfactant, excipient, coloringagent, flavoring agent, preservative, stabilizer, buffering agents,suspending agents, isotonizing agent, binder, disintegrator, lubricant,fluidity promoter, and corrigent. However, the carriers that may beemployed in the present invention are not limited to this list. In fact,other commonly used carriers can be appropriately employed: lightanhydrous silicic acid, lactose, crystalline cellulose, mannitol,starch, carmelose calcium, carmelose sodium, hydroxypropylcellulose,hydroxypropylmethylcellulose, polyvinylacetaldiethylaminoacetate,polyvinylpyrrolidone, gelatin, medium chain fatty acid triglyceride,polyoxyethylene hydrogenated castor oil 60, sucrose,carboxymethylcellulose, corn starch, inorganic salt, and so on.

Furthermore, the present invention provides a method for causingcytotoxicity to cells, which comprises the step of administering theantibody binding to PepT. The antibody binding to PepT has beendescribed above as the antibody binding to PepT contained in the cellgrowth inhibitor of the present invention. The method of this inventioncan be used for treating and preventing disorders caused by cell growth,particularly cancers such as pancreatic cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts graphs showing the results of FACS analyses whichexamined the reactivity of the PepT1 antibody toward the pancreaticcancer cell lines, AsPC-1 and BxPC-3, expressing PepT1 and PepT2 at highlevels, respectively.

FIG. 2 depicts bar graphs showing the results of CDC activitymeasurements of the PepT1 antibody in the AsPC-1 and BxPC-3 cells. Theupper panel shows the CDC activity toward the AsPC-1 cells while thelower panel toward the BxPC-3 cells.

BEST MODE FOR CARRYING OUT THE INVENTION

Herein below, the present invention is described in more detail withreference to Examples.

1. Preparation of Anti-PepT1 Antibody

1-1. Preparation of DEF2A Antibody

Ba/F3 cells expressing the human PepT1 (Ba/F3-PepT1) were washed withPBS and suspended in PBS to a final density of 4×10⁷ cells/ml. This cellsuspension (0.25 ml) was intraperitoneally administered to Balb/c mice(female) for immunization. In a similar manner, immunization wasrepeated at one- to two-week intervals 19 times in total, followed bythe twentieth immunization by administering the cell suspension into thetail vein.

Spleen cells were prepared from these mice, and fused to the mouse P3U1cells by the common method using polyethylene glycol. Resulting cellswere seeded in a 96-well plate, and cultured in a medium containinghypoxanthine, aminopterin, and thymidine (HAT medium) to selecthybridomas. The culture supernatant was recovered on the ninth day fromthe cell fusion, and then screened by ELISA using the germinatingbaculovirus (BV-ELISA) expressing the human PepT1 (PepT1-BV) as anantigen to select for positive wells.

BV-ELISA was performed as follows. That is, PepT1-BV was diluted to be aconcentration of 40 μg proteins/ml in PBS, and distributed in a 96-wellELISA plate (Maxisorp: Nunc) at 100 μl/well. This plate was left atstanding at 4° C. overnight or more, allowing PepT1-BV to adsorb to theplate. Using this plate, ELISA was performed according to the commonmethod.

Hybridomas were cloned by the limiting dilution method using the culturesupernatant from wells judged positive. The culture supernatant ofcloned cells was subjected again to BV-ELISA using the PepT1-BV, and thepositive clone DEF2A was identified.

DEF2A was cultured in an expanded scale, and the culture supernatanttherefrom was examined for the reactivity toward the human pancreaticcancer cell line AsPC-1 by FACS analysis to reveal that the antibodyproduced by the DEF2A clone specifically reacts with AsPC-1 (FIG. 1).

1-2. Preparation of BPT01-13 Antibody

The priming of gp64 transgenic mice (Japanese Patent Application No.2002-180351) was performed by subcutaneous injection of a suspension ofPepT1-BV corresponding to 1 mg protein and 200 ng of pertussis toxin inPBS. Subsequent immunizations were carried out by subcutaneous injectionof a similarly prepared PepT1-BV corresponding to 500 μg protein(containing no pertussis toxin, however). The final immunization wasperformed by injecting PepT1-BV (baculovirus expressing the human PepT1:Japanese Patent Application No. 2002-180351) corresponding to 250 μgprotein into the mouse tail vein. Spleen cells were prepared from thismouse, and fused to the mouse P3U1 cells by the usual method usingpolyethylene glycol.

Screening was performed by FACS using the BaF/3-pepT1 cells.Furthermore, by FACS using the BaF/3-pepT2 cells, the monoclonalantibody “BPT01-13” specifically binding to PepT1 was established.Finally, FACS was performed with AsPC-1 and BxPC-3 cells to confirm thespecific binding to PepT1 on the cancer cells (FIG. 1).

2. CDC Activity Analysis of Anti-PepT1 Antibody

The CDC activity analysis of anti-PepT1 antibody was performed using thePepT1 expression-positive and -negative pancreatic cancer cell lines(AsPC-1 and BxPC-3 cells, respectively).

AsPC-1 cells were cultured in RPMI medium containing 20% FBS, whileBxPC-3 cells in RPMI containing 10% FBS. Cells were seeded on a 96-wellplate (1E4 cells/well) and cultured for two days. ⁵¹Cr (AmershamPharmacia, CJS4) (5 μCi/well) was added to the cells and incubated forone hour to label the cells. After the cells were washed with HAV buffer(300 μl/well), 0.2 μg, 2 μg, or 20 μg/ml anti-PepT antibody (BPT01-13 orDEF2A) was added thereto (100 μl/well), and left at standing on ice for15 min. Then, 100% baby rabbit complement (CEDARLANE, CL3441, Lot. 6213)was added thereto (100 μl/well), and the mixtures were allowed to standat 37° C. for 90 min. After the centrifugation (1,000 rpm, 5 min, 4°C.), the supernatants (100 μl/well) were recovered to measureradioactivity with a gamma counter (Packard Instrument Company,COBRAIIAUTO-GAMMA, MODEL 505). By the following equation, CDC activity(%) was obtained:CDC activity(%)=(A−C)×100/(B−C)wherein A represents the radioactivity in each well; B represents themean radioactivity of the well comprising 2% NP-40 aqueous solution(Nonidet P-40, Nacalai Tesque, 252-23, Lot. M7M7690) (100 μl) instead ofthe complement; and C represents the mean radioactivity of the wellcomprising HAV buffer (200 μl) with neither antibody nor complement.Tests were performed in triplicates to calculate the CDC activity valueand standard error (FIG. 2).

INDUSTRIAL APPLICABILITY

The present inventors have found that antibodies binding to PepT havecytotoxic activity and inhibit cell growth. These antibodies can be usedas a cell growth inhibitor, for example, in treating and preventingcancer.

1. A purified antibody that binds to PepT1, wherein the antibodycontains a human antibody constant region.
 2. The antibody of claim 1,wherein the antibody is not polyclonal.
 3. The antibody of claim 1,wherein the antibody is human.
 4. The antibody of claim 1, wherein theantibody is humanized.
 5. The antibody of claim 1, wherein the antibodyis chimeric.
 6. The antibody of claim 1, wherein the PepT1 is a humanPepT1.
 7. The antibody of claim 1, wherein the antibody binds to anextracellular domain of PepT1.
 8. The antibody of claim 1, wherein theantibody binds to an extracellular domain of human PepT1.
 9. Theantibody of claim 1, wherein the antibody induces antibody-dependentcell-mediated cytotoxic (ADCC) activity when it binds a PepT1-expressingcell.
 10. The antibody of claim 1, wherein the antibody inducescomplement-dependent cytotoxic (CDC) activity when it binds aPepT1-expressing cell.
 11. The antibody of claim 1, wherein the antibodybinds to a PepT1 expression-positive cell but not a PepT1expression-negative cell and wherein the antibody contains a humanantibody constant region.
 12. A method of inhibiting cell growth, themethod comprising providing a purified anti-PepT1 antibody; andcontacting the antibody with a cell that expresses PepT1 on its surface,thereby inducing cytotoxic activity that results in inhibition of growthof the cell.
 13. The method of claim 12, wherein the cell is a humancancer cell and the antibody contains a human antibody constant region.14. The method of claim 12, wherein the cell is a human cancer cell andthe antibody is a human antibody.
 15. The method of claim 12, whereinthe cell is a human cancer cell and the antibody is a humanizedantibody.
 16. The method of claim 12, wherein the cell is a human cancercell and the antibody is a chimeric antibody.
 17. The method of claim12, wherein the cell is a cancer cell.
 18. The method of claim 12,wherein the cell is a pancreatic cancer cell.
 19. The method of claim12, wherein the cell is selected from the group consisting of a livercancer cell, a lung cancer cell, an esophageal cancer cell, a breastcancer cell, and a colon cancer cell.
 20. The method of claim 12,wherein the cytotoxic activity is ADCC activity.
 21. The method of claim12, wherein the cytotoxic activity is CDC activity.
 22. The method ofclaims 12, wherein the antibody is selected from the group consisting ofhuman, humanized, and chimeric.
 23. The method of claim 12, wherein theantibody is not polyclonal.